424. Development of a Scalable Column Chromatography-Based Purification Process To Manufacure Empty Capsid-Free AAV Vectors

Abstract

Recombinant adeno-associated virus (rAAV) has shown great promise as a DNA-based biopharmaceutical agent to treat serious human diseases. In conjunction with efforts in pre-clinical and clinical development, there is a significant need to develop and implement manufacturing processes capable of consistently providing vectors of optimal purity, potency and safety. We have previously reported the development and implementation of a fully scalable rAAV purification process that enabled us to manufacture AAV vectors at a scale to support our clinical development programs. However, like many other reported column-based purification processes, the vectors purified in our second generation purification method contained significant levels of empty capsids. In consideration of the possible deleterious effects of empty capsids, a product-related impurity, on vector efficacy and safety, we have developed a scalable method to separate empty capsids from vector particles. While this separation has been historically achieved using density gradient ultracentrifugation, such methods are not preferred for scalable, cost-effective vector manufacturing. Instead, we have developed a column chromatograpy-based technique to separate empty capsids from DNA containing vector particles. AAV vectors expressing human coagulation factor IX (AAV-hFIX16) were produced using helper virus-free triple transfection of HEK293 cells in roller bottles, and purified using anion exchange chromatography. This empty capsid-containing material was used as the starting point to screen various ion exchange resins to identify conditions that could differentiate between empty and full AAV2 particles. These studies indicated that the surface characteristics of the two particle types are very similar. However, identification of subtle, pH-dependent differences in binding to selected resins, in conjunction the selective use of chaotropic agents, allowed us to separate these two populations effectively. Our data indicated that more than 90% of the empty particles were removed from the purified vector using this newly developed column chromatography technique, with approximately 60% recovery of the DNA-containing vector particles. This new column step, in combination with the column-based scalable method that we previously reported, now enables us to efficiently manufacture empty capsid-free recombinant AAV using a fully scalable process.